Sugar derivative

ABSTRACT

For extending the uses of a compound represented by, Chemical formula 1, the object of the present invention is to provide a derivative of cyclic tetrasaccharide whose physicochemical properties are changed from those of cyclic tetrasaccaride, a composition comprising the same, and a process for producing the same. The present invention solves the above object by providing a derivative of cyclic tetrasaccharide, which is produced by the steps of reacting a compound represented by Chemical formula 1 with a reactive reagent and substituting one ore more hydroxyl groups with substituents except hydroxyl group and O-glycosyl group; a composition comprising the same; and a process for producing the derivative of cyclic tetrasaccharide.  
     Chemical formula 1

TECHNICAL FIELD

[0001] The present invention relates to novel derivatives of cyclictetrasaccharide, compositions comprising the same, and processes forproducing the derivatives of cyclic tetrasaccharide.

BACKGROUND ART

[0002] A cyclic tetrasaccharide, having a structure composed of fourglucose molecules bound together via alternating α-1,3-glucosidiclinkage and α-1,6-glucosidic linkage, i.e., a compound having astructure ofcyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1-},represented by Chemical formula 1 (hereinafter, briefly called as“cyclotetrasaccharide”), and a process for producing the saccharide byallowing a hydrolytic enzyme, alternanase, to act on a polysaccharide,alternan, were reported by Gregory L. Cote et al. in European Journal ofBiochemistry, Vol. 226, pp.641-648, 1994. However, the industrialproduction of cyclotetrasaccharide by the above process has beendifficult because of the expensiveness of material alternan. Recently,as disclosed in International Publication Nos. WO 01/90,338(international application No. PCT/JP01/04,276) and WO 02/10,361(international application No. PCT/JP01/06,412), applied for by the sameapplicant as the present invention; a process for producingcyclotetrasaccharide, using both enzymes originated from microorganismsand inexpensive starch as a material, was established. Therefore, theprocess enables us to produce cyclotetrasaccharide industrially, and theuses of the saccharide are now developing.

[0003] Chemical formula 1

[0004] In order to extend the uses of cyclotetrasaccharide, it ismeaningful to produce novel derivatives of cyclotetrasaccharide, havingdifferent physical properties from those of cyclotetrasaccharide. Asreports referred to the derivatives of cyclotetrasaccharide, JapanesePatent Application No. 67,282/2001 (title of the invention, “Branchedcyclic tetrasaccharide, its preparation and uses”) and U.S. Pat. No.5,889,179 disclosed the derivative having a structure ofglucosyl-cyclotetrasaccharide, i.e., branched cyclotetrasaccharide.Since the derivatives of cyclotetrasaccharide described in those areproduced by binding O-glycosyl groups with cyclotetrasaccharide by usingO-glycosyl group-transferring enzymes, the resulting derivatives haveonly O-glycosyl groups. Therefor, it is restricted to change thephysical properties of cyclotetrasaccharide by binding an O-glycosylgroup(s) with cyclotetrasaccharide.

[0005] The object of the present invention is to provide derivatives ofcyclotetrasaccharide, having different physical properties from those ofcyclotetrasaccharide, compositions comprising the same, and processesfor producing the derivatives of cyclotetrasaccharide.

DISCLOSURE OF INVENTION

[0006] In the course of extensive studies, the present inventors newlyfound that derivatives of cyclotetrasaccharide represented by Formula 1,cyclotetrasaccharide whose hydroxyl groups were substituted by othersubstituents, can be obtained by allowing reactive reagents to act oncyclotetrasaccharide without using enzymatic reaction system withgenerally known various saccharide-related enzymes such as cyclodextringlucanotransferase, α-galactosidase, β-galactosidase, lysozyme, otherglycosyltransferases, other saccharide hydrolases, and saccharidephosphorylases. The present inventors found that various substituentscould be specifically introduced into cyclotetrasaccharide in additionto O-glycosyl group or modified O-glycosyl group, which could beintroduced by conventional enzymatic systems, and physical properties ofcyclotetrasaccharide could be arbitrarily changed by the above reaction,and accomplished the present invention. According to the presentinvention, physical properties of cyclotetrasaccharide such assweetness, less fermentability, low cariogenicity, low caloric property,osmotic pressure-controlling property, excipiency, gloss-impartingproperty, moisture-retaining property, viscosity, syneresis-preventingproperty, solidification-preventing property, inclusion complex-formingproperty, flavor-keeping property, stability, property for preventingthe crystallization of other saccharide,starch-retrogradation-preventing property,protein-denaturation-preventing property, lipid-deterioration-preventingproperty, stability to acids, non-aminocarbonyl-reactivity,dielectricity, polarizability, and electric-conductivity can bearbitrarily changed. For example, a saccharide derivative, having astrong hydrophobic substituent(s), has a lipophilic property which isnot inhered to intact cyclotetrasaccharide. Since a derivative ofcyclotetrasaccharide, having a highly reactive substituent, has asuperior binding ability with cyclotetrasaccharide or other organiccompounds, it can be advantageously used for polymerizingcyclotetrasaccharides or binding with other organic compounds. Thephysical properties of other organic compounds can be changed byallowing the organic compounds to bind with cyclotetrasaccharide byusing the above derivatives.

[0007] Formula 1

[0008] In the above formula, R₁ to R₁₂ mean optional substituents, butone or more of them are those except hydroxyl group and O-glycosylgroup.

[0009] The present invention solves the objects by the above method. Thepresent invention provides the derivatives of cyclotetrasaccharide whosephysical properties such as lipophilic property and binding property arechanged from those of cyclotetrasaccharide by substituting hydroxylgroups of cyclotetrasaccharide with other substituents except hydroxylgroups and O-glycosyl groups.

[0010] The present invention also provides the uses of the derivativesof cyclotetrasaccharide whose physical properties such as lipophilicproperty and binding property are changed from those ofcyclotetrasaccharide.

[0011] The present invention further provides processes for producingthe derivatives of cyclotetrasaccharide whose physical properties suchas lipophilic property and binding property are changed from those ofcyclotetrasaccharide.

BEST MODE FOR CARRYING OUT THE INVENTION

[0012] A derivative of cyclotetrasaccharide as referred to as in thepresent invention means a derivative produced by substituting one ormore hydroxyl groups of cyclotetrasaccharide with other substituentsexcept hydroxyl groups and O-glycosyl groups by reacting a reactivereagent with cyclotetrasaccharide (hereinafter, briefly called “aderivative of cyclotetrasaccharide”). An O-glycosyl group as referred toas in the present invention means O-glycosyl group which can besubstituted with hydroxyl group of cyclotetrasaccharide, and thatmodified in an acceptable range of being substituted by an enzymaticreaction system. A reaction system, used for producing the derivativesof cyclotetrasaccharide of the present invention, is a chemical reactionsystem using a reactive reagent and not an enzymatic reaction system.Therefore, it is possible to introduce modified O-glycosyl groups, whichare hardly introduced by conventional enzymatic reaction systems, intocyclotetrasaccharide. As chemical reactions, esterification,etherification, sulfonation, amination, etc., which are conventionallyused for producing derivatives of saccharide such as starch, can beused. Specifically, methods for chemically modifying cyclodextrins,cyclic saccharides constructed by 6-8 glucose molecules, can be widelyused. For example, such methods described in Joshua Boger et al.,Helvetica Chimica Acta, Vol. 61, pp.2190-2218, 1978; C. K. Lee,Development in Food Carbohydrate, pp.1-89, 1980, published by AppliedScience Publishers; K. Yoshimoto et al., Chemical and PharmaceuticalBulletin, Vol. 30, No. 4, 1, pp.1169-1174, 1982; Carbohydrates asOrganic Raw Material, 1991, published by VCH; and Methods inCarbohydrate Chemistry II, 1963, published by Academic Press; are allusable in the present invention and can be arbitrarily selectedaccording to the objective derivative of cyclotetrasaccharide.

[0013] A hydrocarbon group as referred to as in the present inventionmeans a group constructed by one or more carbon atoms and hydrogenatoms, and includes saturated and unsaturated hydrocarbon groups. Forexample, aliphatic hydrocarbon groups having carbon number 1-18 such asmethyl, ethyl, ethynyl, propyl, isopropenyl, 1-propenyl, I-propynyl,2-propenyl, butyl, isobutyl, s-butyl, t-butyl, vinyl, 1,3-butadienyl,2-butenyl, pentyl, isopentyl, neopentyl, t-pentyl, 1-methylpentyl,2-methylpentyl, 2-pentenyl, 2-penten-4-ynyl, hexyl, isohexyl,5-methylhexyl, heptyl, and octyl; aliphatic cyclic hydrocarbon groupssuch as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, andcyclohexenyl; mono- or polycyclic aromatic hydrocarbon groups having abenzene ring as a basic frame such as phenyl and naphthyl; can be listedas hydrocarbon groups.

[0014] A substituent, having oxygen except hydroxyl group and O-glycosylgroup, as referred to as in the present invention means everysubstituent having oxygen except hydroxyl group and O-glycosyl group andusually means substituents constructed by oxygen atom and other atoms:for example, hydrogen, carbon, nitrogen, sulfur, halogen, etc. Forexample, ester form of saturated, unsaturated, branched or linear fattyacids such as caproic acid, caprylic acid, capric acid, lauric acid,myristic acid, paltmitic acid, stearic acid, arachic acid, behenic acid,lignoceric acid, zoomaric acid, oleic acid, linoleic acid, linolenicacid, gadoleic acid, erucic acid, and selacoleic acid; ester form ofcarboxylic acid such as acetic acid (acetyl), propionic acid(propionyl), and benzoic acid (benzyl); sulfuric ester, phosphoricester; ether form of alkyl alcohol having carbon number of 1-18 (alkoxylgroup) such as methanol (methoxy), ethanol (ethoxy), and propanol(propoxy); ether form of aromatic alcohol such as benzyl alcohol andphenol; functional group having oxygen atom except hydroxyl group suchas carboxyl, aldehyde, ketone; hydrocarbon group having functionalgroup(s) such as carboxyl, aldehyde, keto, and hydroxyl; and variousoxide.

[0015] A substituent having nitrogen as referred to as in the presentinvention means every substituent having one or more nitrogen atoms andusually means a substituent constructed by nitrogen atom(s) and otheratoms: for example, a functional group such as amino, hydroxylamino,oxime, carbamide, carbamic acid ester, thiocarbamic acid O-ester,thiocarbamic acid S-ester, imino, azirine, nitro, nitroso, azide, diazo,nitrile, isonitrile, cyano, isocyanate, isothiocyanate, and cyanuricchloride; substituents having those; and various nitrogen compounds.

[0016] A substituent having sulfur as referred to as in the presentinvention means every substituent having one or more sulfur atoms andusually means a substituent constructed by sulfur atom and other atoms:for example, a functional group such as mercapto, sulfonyl, sulfonicacid, sulfide, disulfide, sulfoxide, sulfonyl imido, sulfen, sulfine,thiolsulfinato, thiolsulfonato, sulfilimine, and p-toluene sulfonyl;substituent having those; and various sulfur compounds.

[0017] A substituent having halogen as referred to as in the presentinvention means every substituent having one or more halogen atoms, andusually means a substituent constructed by halogen atom(s) and otheratoms: for example, functional group such as fluorine, chlorine,bromine, and iodine; substituents having those; and various halide.

[0018] A derivative of cyclotetrasaccharide of the present invention canbe produced by the steps of dissolving, suspending or soakingcyclotetrasaccharide in a solvent mentioned after, adding a reactivereagent as a substituent donor, if necessary, with a catalyst, andreacting under suitable conditions (such as temperature, time, pH, andpressure) while mixing or stirring by a suitable method. The resultingderivative of cyclotetrasaccharide can be purified by removing remainingreactive reagent, solvent and/or catalyst using suitable purificationprocedures.

[0019] For example, a hydrocarbon solvent such as propane, butane,pentane, hexane, isohexane, heptane, isoheptane, isooctane, benzine,rubber volatile oil, soybean volatile oil, mineral spirit, cleaningsolvent, petroleum ether, petroleum benzine, ligroin, kerosine,cyclohexane, methylcyclohexane, benzene, benzol, toluene, toluol,xylene, xylol, ethylbenzene, cumene, mesitylene, light-solvent naphtha,heavy-solvent naphtha, tetrarine, decaline, creosote oil, and turpentineoil; a halogen solvent such as methyl chloride, methylene chloride,chloroform, carbon tetrachloride, dichloro-difluoro-methane, ethylchloride, 1,2-dichloroethane, 1,2-dibromoethane, tetrachloroethane,dichloroethylene, trichloroethylene, perchloroethylene, dichloropropane,amyl chloride, dichloropentane, monochlorobenzene, o-dichlorobenzen,trichlorobenzene, and bromobenzene; alcoholic and phenolic solvent suchas methanol, ethanol, n-propylalcohol, isopropylalcohol, n-butylalcohol,isoamylalcohol, synthetic amylalcohol, fusel oil,methylisobutylcarbinol, n-hexylalcohol, 2-ethylbutanol, n-octylalcohol,2-ethylhexanol, cyclohexanol, furfurylalcohol,tetrahydrofurfurylalcohol, benzylalcohol, phenol, and cresol; ethersolvent such as ethylether, isopropylether, n-butylether,dichloroethylether, anisole, dioxane, tetrahydrofurane,tetrahydropyrane, and benzylethylether; acid and their esters such asformic acid, acetic acid, acetic anhydride, butyric acid, methylformate, ethyl formate, butyl formate, amyl formate, methyl acetate,ethyl acetate, isopropyl acetate, butyl acetate, s-butyl acetate, amylacetate, isoamyl acetate, 2-ethylhexyl acetate, cyclohexyl acetate,ethyl propionate, butyl propionate, amyl propionate, butyl butyrate,diethyl carbonate, diethyl oxalate, methyl lactate, ethyl lactate,triethylphosphate, γ-butyrolactone, and trifluoroacetic acid; apolyalcohol, its ether and ester such as ethylene glycol, ethyleneglycol monomethyl ether, ethylene glycol monoethyl ether, ethyleneglycol monobutyl ether, ethylene glycol monoacetate, ethylene glycolmonomethyl ether acetate, ethylene glycol monoethyl ether acetate,ethylene glycol dimethyl ether, diethylene glycol, diethylene glycolmonomethyl ether, diethylene glycol monoethyl ether, diethylene glycoldimethyl ether, triethylene glycol, propylene glycol, hexylene glycol,and glycerin; aldehyde, acetal, and ketone such as furfural, methylal,acetal, acetone, methylethylketone, methylisobutylketone,diisobutylketone, methyl oxide, acetylacetone, diacetonealchol,cyclohexanone, methylcyclohexanone, and isophorone; a nitrogen compoundsuch as nitromethane, nitroethane, 1-nitro-propane, 2-nitro-propane,nitrobenzene, acetonitrile, diethylamine, triethylamine,cyclohexylamine, ethylene diamine, aniline, pyridine, picoline,quinoline, monoethanolamine, diethanolamine, morpholine, dimethylformamide, dimethylacetoamide, hexamethyl phospholtriamide, and N-methylpyrrolidone; a sulfuric compound such as anhydrous sulfide, carbondisulfide, thiophene, sulfolane, and dimethylsulfoxide; can be used as asolvent in the present invention. Among those, it is preferable to use asolvent, which can solubilize either cyclotetrasaccharide or aderivative of cyclotetrasaccharide of the present invention, to increasethe efficiency of the synthesis. In the case of being preferable toreact under anhydrous condition, it is desirable to remove moisture by asuitable dehydrating agent. In the case of being acceptable to containwater in the reaction system, water can be used as a solvent. Thosesolvents can be used alone or along with one or more other solvents bymixing.

[0020] Cyclotetrasaccharide usable in the present invention is notrestricted to its origin and a process for producing it as long as notaffecting the object of the present invention. Cyclotetrasaccharide,which is conventionally obtainable by allowing alternanase to act onalternan, and one which is obtainable by the method disclosed inInternational Publication No. WO 02/10,361 (international applicationNo. PCT/JP01/06,412), i.e., an enzymatic method of allowingα-isomaltosyl-transferring enzyme andα-isomaltosylglucosaccharide-forming enzyme to act on starch, can beused. In the case of producing a derivative of cyclotetrasaccharide inan industrial level, the later method described above is advantageouslyused because cyclotetrasaccharide can be obtained in lower cost. Varyingdepending on the kind of objective derivative and the reaction system, adesirable concentration of cyclotetrasaccharide in a solvent can beselected from the range of, usually, 0.1-80% (w/v), desirably, 0.1-20%(w/v).

[0021] α-Isomaltosyl-transferring enzymes, disclosed in InternationalPublication No. WO 01/90,338 (international application No.PCT/JP01/04,276), and α-isomaltosylglucosaccharide-forming enzymes,disclosed in International Publication No. WO 02/10,361 (internationalapplication No. PCT/JP01/06,412), can be used for producingcyclotetrasaccharide from starch in an industrial scale. Both aboveenzymes can be prepared by cultivating Bacillus globisporus C9 (FERMBP-7143), Bacillus globisporus C11 (FERM BP-7144), Bacillus globisporusN75 (FERM BP-7591), Arthrobacter globiformis A19 (FERM BP-7590), orArthrobacter ramosus S1 (FERM BP-7592) according to the method describedin above specifications. Both above enzymes prepared from above strainshave physicochemical properties shown in Table 1. Cyclotetrasaccharidecan be produced from starch material with a satisfactory yield by usingabove enzymes in combination. The cyclotetrasaccharide content of thepreparation, obtained by the method described in above specifications,is usually about 60% (w/w) (hereinafter, “% (w/w)” is brieflyabbreviated as “%”, unless specified otherwise) or higher, and thesaccharide can be used for producing the derivative ofcyclotetrasaccharide of the present invention. In the case of desiring apreparation having a relatively high cyclotetrasaccharide content, highcyclotetrasaccharide content fractions can be collected by applying thelow cyclotetrasaccharide content preparation to a strong-acid cationexchange resin (salt form) column chromatography with fixed-bed, movingbed, or semi-moving bed. The high cyclotetrasaccharide contentfractions, thus obtained, contain cyclotetrasaccharide in a content ofabout 98%, on a dry solid basis, and can be used as materials forproducing a derivative of cyclotetrasaccharide of the present invention.TABLE 1 α-Isomaltosyl- α-Isomaltosyl- glucosaccharide- transferringenzyme forming enzyme Molecular About 82,000-136,000 About74,000-160,000 weight daltons daltons (Analytical (SDS-gelelectrophoresis) (SDS-gel electrophoresis) method) Isoelectric about3.7-8.3 about 3.8-7.8 point (pI) (Analytical Ampholine electrophoresisAmpholine electrophoresis method) Optimum about 45-50° C. about 45-60°C. or temperature about 45-65° C. (in the presence of 1 mM Ca²⁺)(Condition) Reaction at pH 6.0 Reaction at pH 6 or for 30 min pH 8.4 for60 min Optimum pH pH about 5.5-6.5 pH about 6-8.4 (Condition) Reactionat 35° C. Reaction at 35° C. for 30 min for 60 min Thermal Stable up toStable up to about 55° C. stability about 45° C. or about 60° C. (in thepresence of 1 mM Ca²⁺) (Condition) Incubation at pH 6.0 Incubation at pH6 or for 60 min pH 8 for 60 min pH Stability Stable in a range of Stablein a range of pH about 3.6-10.0 pH about 4.5-10.0 (Condition) Incubationat 4° C. Incubation at 4° C. for 24 hours for 24 hours

[0022] In the case of selecting the reaction system not preferringmoisture as a process for producing the derivative ofcyclotetrasaccharide in the present invention, it is preferable to use apreparation of cyclotetrasaccharide, comprising moisture as low aspossible, for increasing the reaction yield. Therefore,cyclotetrasaccharide in a solid or powdery form, having low moisturecontent, produced by suitable concentrating and drying process, is usedin such a case. Since crystalline cyclotetrasaccharide may have five tosix molecules of water per molecule even in a solid or powdery form, itis desirable to use anhydrous crystalline and anhydrous amorphouscyclotetrasaccharide, which are disclosed in International PublicationNos. WO 01/90,338 (international application No. PCT/JP01/04,276) and WO02/10,361 (international application No. PCT/JP01/06,412) and JapanesePatent Application No. 10,991/2001, for producing the derivative ofcyclotetrasaccharide of the present invention. Preferable moisturecontent of cyclotetrasaccharide is 3% or lower, more preferably, 1% orlower.

[0023] When material cyclotetrasaccharide can not be dissolvedsufficiently because of using a solvent which can hardly or not dissolvethe saccharide, it is desirable to use powdery cyclotetrasaccharide forincreasing the reaction efficiency. The diameter of granule of powderycyclotetrasaccharide should be controlled to a suitable size to thesolvent and the reaction condition. Usually, granule having the smallerdiameter gives the faster reaction rate. Therefore, the reaction ratecan be controlled by choosing the diameter of the granule. The diameterof powdery cyclotetrasaccharide, used in the present invention, can bearbitrarily determined according to the objective derivative or reactionsystem, and is controlled to be, usually, 500 μm or lower, preferably,0.1-250 μm, more preferably, 1-100 μm.

[0024] Lewis acids such as aluminum chloride, aluminum bromide, zincchloride, antimony chloride, boron fluoride, copper chloride, tinchloride, and phosphorus chloride; Brφnsted acids such as hydrogenfluoride and phosphoric acid; hydroxide or oxide of alkaline metal oralkaline earth metal such as sodium hydroxide, potassium hydroxide,magnesium hydroxide, calcium hydroxide, magnesium oxide, and potassiumoxide; bases such as basic organic compounds including amines; and heavymetals such as platinum, palladium, nickel, cobalt, copper, chromium,molybdenium, silver, zinc and their oxide, sulfide, and Raney catalystcan be used as catalysts in the present invention, and one or morecatalysts can be used in combination. These catalysts can be optionallyselected in accordance with the objective derivative ofcyclotetrasaccharide and the reaction system. The amount of catalyst ispreferable to be, usually, 0.0001% or higher, desirably, 0.001-10,000%,more desirably, 0.01-1,000% to the weight of cyclotetrasaccharide.

[0025] The reactive reagent as referred to as in the present inventionmeans one or more reagents selected from the group consisting of acids,bases, alcohols, aldehydes, ketones, halogens, amines, cyanogens,nitrites, oxiranes, isocyanates, isothiocyanates, thiols, sulfones andtheir reactive derivatives, introducing hydrocarbon group, substituentshaving oxygen, those having nitrogen, those having sulfur, and thosehaving halogen into cyclotetrasaccharide. The molar ratio of thereactive reagent to cyclotetrasaccharide can be suitably determined inaccordance with the objective derivative of cyclotetrasaccharide and thereaction system, and selected from the range of, usually, 0.01-10,000mole %, desirably, 0.1-1,000 mole %.

[0026] Reaction conditions such as reaction temperature, reaction time,and reaction pressure can be suitably selected in accordance with theobjective derivative of cyclotetrasaccharide and the reaction system.Generally, it is preferable to set reaction temperature to a relativelyhigh one in the case of endothermic reaction and a relatively low one inthe case of exothermic reaction. The reaction temperature can be usuallyselected from the range of that proceeding the reaction substantially,desirably, −50 to 200° C. The reaction time can be usually selected fromthe range of that until accomplishing the reaction, desirably, oneminute to 100 hours. The reaction can be carried out, usually, undernormal pressure, and, optionally, under pressurized or reduced pressurecondition. Optionally, the reaction can be accelerated by irradiatingthe reaction mixture with lights such as ultraviolet rays, visible rays,and infrared rays; radiations such as X-rays and γ-rays; andelectromagnetic radiation.

[0027] A reaction product comprising the derivative ofcyclotetrasaccharide of the present invention, thus obtained, can beusually used intact after removing remaining reactive reagents and/orsolvent by the methods such as filtration, extraction, separating,separatory precipitation, dialysis, and distillation. In the case ofrequiring the derivative of cyclotetrasaccharide, having a relativelyhigh purity, the reaction product can be purified by conventionalmethods for purifying saccharides or saccharide derivatives such asthin-layer chromatography, column chromatography, ion-exchangechromatography, high performance liquid chromatography, distillation,and crystallization. Those purification methods can be optionally andarbitrarily used in combination.

[0028] A reaction system using a solvent can be usually selected forproducing the derivatives of the present invention industrially. In somecases, other reaction system can be used as in the case ofcyclodextrins. For example, co-precipitation method, slurry method,paste method, and dry-mixing method, described in Allan R. Hedges,Chemical Reviews, Vol.98, 1998, can be used.

[0029] The followings explain representative processes for producing thederivatives of cyclotetrasaccharide of the present invention.

[0030] <1> Esterification and etherification

[0031] Esterification and etherification can be used for introducinghydrophobic groups such as alkyl group and aromatic hydrocarbon groupinto cyclotetrasaccharide. Esters constructed by cyclotetrasaccharideand a carbonic acid such as acetic acid and benzoic acid can be obtainedby reacting cyclotetrasaccharide with corresponding acid anhydrides oracid halides in a basic solvent such as pyridine. Esters constructed bycyclotetrasaccharide and sulfuric acid can be obtained by reactingcyclotetrasaccharide with a complex of sulfur trioxide and dimethylsulfoxide or pyridine in a flow current of inert gas or rare gas. Estersconstructed by cyclotetrasaccharide and fatty acids such as caproicacid, caprylic acid, capric acid, lauric acid, myristic acid, paltmiticacid, stearic acid, arachic acid, behenic acid, lignoceric acid,zoomaric acid, oleic acid, linoleic acid, linolenic acid, gadoleic acid,erucic acid, and selacoleic acid can be obtained by condensingcyclotetrasaccharide with those fatty acids in the presence of a basiccatalyst or reacting cyclotetrasaccharide with corresponding fatty acidhalides.

[0032] <2> Sulfonylation

[0033] Sulfonylated derivatives of cyclotetrasaccharide are useful asreaction intermediates for synthesizingvarious derivatives. Afterintroducing tosyl (4-methylbenzenesulfonyl) group, mesyl(methanesulfonyl) group, or other related allylsulfonyl groups intocyclotetrasaccharide, substituents such as amino group, azido group,halogen group can be introduced into the resulting derivatives bynucleophilic displacement reaction. The position which should beintroduced substituent can be restricted by selecting the reactioncondition. For example, tosylated cyclotetrasaccharide,cycrotetrasaccharide whose primary hydroxyl groups are tosylated, can beobtained by reacting cyclotetrasaccharide with tosyl chloride inpyridine. Ferrocenecarbonyl cyclotetrasaccharide can be produced byintroducing ferrocenecarbonyl group into cyclotetrasaccharide byallowing the saccharide to react with sodium ferrocenecarbonate indimethylsulfoxide and heating. The reaction can be carried out accordingto the method described in Akihiko Ueno et al., MacromolecularChemistry, Rapid Communications, Vol.6, pp.231-233, 1985, and IwaoSuzuki et al., Bulletin of the Chemical Society of Japan, Vol.66,pp.1472-1481, 1993. Amino cyclotetrasaccharide can be produced by thesteps of diazotizing above tosyl-cyclotetrasaccharide with sodium azideand reducing the resulting derivative to convert the diazo group intoamino group (J. Boger et al., Helvetica Chimica Acta, Vol. 61, pp.2190,1978). On the other hand, secondary hydroxyl groups ofcyclotetrasaccharide are tosylated by reacting cyclotetrasaccharide withm-nitrophenyltosylate at 60° C. in dimethylformamide-alkaline aqueoussolution (Akihiko Ueno et al., Tetrahedron Letters, Vol.23, pp.3451,1982). S-Alkyl derivative of cyclotetrasaccharide can be produced by thesteps of epoxidizing tosyl-cyclotetrasacchride under an alkalinecondition and reacting the resultant with alkyl sulfide to introducealkyl group via sulfide bond into cyclotetrasaccharide (R. Breslow etal., Journal of the American Chemical Society, Vol.105, pp.1390, 1983;Murakami et al., Chemistry Letter, pp.553, 1988; and Ikeda et al.,Tetrahedron Letters, Vol.31, pp.5045, 1990).

[0034] <3> 2,2,6,6-Tetramethyl-1-piperidinyloxilation

[0035] This process can be used for producing carboxylatedcyclotetrasaccharide. Carboxylated cyclotetrasaccharide can be obtainedby oxidizing primary hydroxyl groups of cyclotetrasaccharide to formcarboxyl group by the steps of mixing cyclotetrasaccharide with2,2,6,6,-tetramethyl-1-pipetidinyloxi-sodium perchlorate, sodiumbromide, and sodium chlorite and reacting at pH 9-11. Carboxyl group canbe bound with compounds having amino group via amide bond. The reactioncan be carried out according to the method described in Ichikawa et al.,Tetrahedron: Asymmetry, Vol.11, pp.389-392, 2000.

[0036] <4> Polymerization

[0037] This process can be used for polymerizing cyclotetrasaccharides.Polymerized cyclotetrasaccharide in a gel form can be obtained byreacting cyclotetrasaccharide with epichlorohydrin in alkaline solutionfor cross-linking. The reaction can be carried out according to themethods described in Jozsef Szejtli et al., Journal of MaterialsChemistry, Vol.7, No.4, 1997; Akira Hamada, Modular Chemistry, edited byJ. Michl, pp.361-370, 1997; and Komiyama, “Kagaku (Science)” (inJapanese), Vol.59, No.2, pp.105-112, 1989.

[0038] <5> Introduction of various functional groups

[0039] Hydroxyl groups of cyclotetrasaccharide can be substituted withother functional groups. Derivatives of cyclotetrasaccharide, having areactive functional group, can be bound with other organic compounds. Analdehyde group(s) can be introduced into cyclotetrasaccharide, forexample, by the steps of introducing methyl halide group intocyclotetrasaccharide and oxidizing the resultant with dimethylsulfoxideor hexamine. A halogen group(s) can be introduced intocyclotetrasaccharide: for example, a chloro-group(s) can be introducedinto cyclotetrasaccharide by the steps of mixing the saccharide withconcentrated hydrochloric acid and zinc chloride and heating or allowingthem to react in a flow current of dried hydrochloric acid gas. An aminogroup(s) can be introduced into cyclotetrasaccharide by the steps ofallowing the saccharide to react with halogenocarboxylic acid halide orepichlorohydrin to produce cyclotetrasaccharide halide and allowing theresultant to react with ammonia. A mercapto-group(s) can be introducedinto cyclotetrasaccharide by the steps of allowing the abovecyclotetrasaccharide halide to react with sulfurating agent such assodium thiosulfate and reducing the resultant with lithium aluminumhydride.

[0040] The derivatives of cyclotetrasaccharide of the present inventioncan be bound with other substances via their functional groups.Therefore, they can be bound with biologically and physiologicallyactive substances: for example, cytokines such as interferon, tumornecrosis factor, erythropoietin, and interleukin 2; hormones such asinsulin and steroid; and amino acids, oligopeptides, polypeptides,proteins, nucleic acids, saccharides, lipids, vitamins, and antibiotics.Also, since they can be bound with coloring materials and fluorescentsubstances such as dansylgycine, N,N′-dimethylaminobenzoyl group, methylred, paramethyl red, anthracene-9-carbonyl group, pyrene, andazobenzene, they can be used as reagents for the detection. Furthermore,since they can be bound with 2-hydroxypropyl group, pyridoxamine group,p-methoxyphenol, p-nitrophenol, benzofuroxan, and metaphorbol, they canbe used as reaction catalysts for other substances. Also, since they canbe bound with high molecular carrier such as polyvinylalcohol,polyacrylamide, polyethyleneglycol, polypropyleneglycol,polymethylvinylether, cellulose, and their derivatives, they can be usedfor analyzing or purifying other substances.

[0041] Cyclotetrasaccharide provides twelve non-anomeric hydroxyl.groups as reactive functional groups for usual substituting reactions.This means that a composition containing derivatives ofcyclotetrasaccharide, which have different degree of substitution, invarious proportions would be formed depending on the kinds and theconditions of the reaction. The degree of substitution can be controlledby controlling time, temperature, and reagent concentration of thereaction. For avoiding the substitution by later reaction, adequateprotection groups can be introduced in advance intocyclotetrasaccharide. The degree of substitution can be conventionallydetermined by measuring the integral values of ¹H-NMR or byspectroscopic procedures. The derivatives of cyclotetrasaccharide of thepresent invention are not restricted by their degrees of substitution.They having various degree of substitution can be used according totheir uses. Usually, derivatives having an average degree ofsubstitution of one or more can be preferably used. Sincecyclotetrasaccharide has two primary hydroxyl groups and ten secondaryhydroxyl groups, substituents can be selectively introduced by usingtheir subtly difference of reactivities.

[0042] The derivatives of cyclotetrasaccharide, which are obtained bythe process of the present invention, can be widely used in variousfields such as catalyst, fiber, wrapping, construction, paint, analysis,electrical engineering, and correspondence as well as food industry,cosmetic industry, and pharmaceutical industry. The derivatives ofcyclotetrasaccharide of the present invention; which are introducedhydrophobic groups such as phenyl group, alkyl group, and acetyl group;are lipophilic and useful as surfactants for foods, cosmetics, andpharmaceuticals. The derivatives, introduced sulfate ester, can beadvantageously incorporated into cosmetics as a good moisture-retainingagent or skin care agent. The derivatives, introduced functional groupshaving a binding ability such as vinyl group, amino group, carboxylgroup, mercapto group, and halogen group, or substituents having thosefunctional groups, can be bound with other organic compounds and/orcyclotetrasaccharide. Therefore, they can be used for producing novelorganic compounds by forming polymers as well as homo- and heterodimers;producing supports for analysis, detection, and purification by bindingwith macromolecular supports; changing properties of other compounds aswell as proteins; and reacting with other compounds in the presence ofcatalysts. The derivatives, introduced functional groups such as cyanogroup, nitro group, and nitroso group or substituents having thosefunctional groups, can be used to pharmaceuticals as antibacterialagent, immunity activating agent, and anticancer agent; and to materialsfor fibers, wrappings, and constructions. Since the derivatives ofcyclotetrasaccharide of the present invention, having a dissociativefunctional group such as carboxyl group, halogen group, etc., and theyhaving a polarizing functional group such as hydroxyl group, aminogroup, etc., have a dielectric property; they can be used as additivesfor fuel battery. The derivative of cyclotetrasaccharide of the presentinvention, having an alkyl alcohol group such as hydroxymethyl group,hydroxyethyl group, hydroxypropyl group, etc., shows a higher solubilityin water than that of cyclotetrasaccharide. The derivatives ofcyclotetrasaccharide of the present invention, having two or morefunctional groups and/or two or more kinds of functional groups, can beused as a cross-linking agent.

[0043] Since the derivatives of cyclotetrasaccharide of the presentinvention have a structure of cyclotetrasaccharide as a basic frame,they usually have the properties and functions of cyclotetrasaccharidepartially. Therefore, they can be used for the similar object withcyclotetrasaccharide. They can be used according to the uses ofcyclotetrasaccharide, which are disclosed in International PublicationNos. WO 01/90,338 (international application No. PCT/JP01/04,276) and WO02/10,361 (international application No. PCT/JP01/06,412), applied forby the same applicant as the present invention. The following outlinesthe uses of the derivatives of cyclotetrasaccharide of the presentinvention.

[0044] The derivatives of cyclotetrasaccharide of the present inventionusually have the properties and functions of cyclotetrasaccharidepartially though they are varied depending on the substituents. In manycases, such derivatives show a low or non-sweetness and a satisfactorytaste, and are non-reducing and stable saccharides. Therefore, when theyare admixed with other materials, especially, amino acids and substancesconstructed by amino acids such as oligopeptides and proteins, andprocessed, the mixture would not turn brownish and not form strangesmells, and other materials mixed would not be damaged. Therefore, thederivatives of cyclotetrasaccharide of the present invention can be usedas materials in various fields such as foods, cosmetics, andpharmaceuticals.

[0045] Since the derivatives of cyclotetrasaccharide of the presentinvention are constructed with cyclotetrasaccharide as a basic frame,they are substantially hardly hydrolyzed by amylases and α-glucosidases.When they are orally ingested, they are not assimilated and hardlyfermented by intestinal bacteria. Therefore, they can be used aswater-soluble dietary fibers having an extremely low calorie. Also,since the derivatives of cyclotetrasaccharide of the present inventionare hardly fermented by dental caries-inducing bacteria, they can beused as sweeteners hardly inducing dental caries. Further, they alsohave a function of preventing the adhesion and solidifying of solids inoral. The derivatives of cyclotetrasaccharide of the present inventionexhibit various properties such as osmosis-controlling ability, fillingability, gloss-imparting ability, moisture-retaining ability, viscosity,ability of preventing the crystallization of other saccharides, and lessfermentability. The derivatives of cyclotetrasaccharide of the presentinvention may be harmful and poisonous depending on the kind ofsubstituents. If the derivatives of cyclotetrasaccharide of the presentinvention and the compositions comprising the same are confirmed to besafe, they can be advantageously used to various compositions such asfoods, tobacco, cigarette, feeds, pet foods, cosmetics, andpharmaceuticals as seasoning, taste-improving agent, quality-improvingagent, stabilizer, discoloration-preventing agent or filler.

[0046] The derivatives of cyclotetrasaccharide of the present inventionand the compositions comprising the same can be incorporated into foods,cosmetics, and pharmaceuticals along with other ingredients which areusually used for foods, cosmetics, and pharmaceuticals. The followingshows the other ingredients concretely.

[0047] The derivatives of cyclotetrasaccharide of the present inventionand the compositions comprising the same can be admixed with variousfats: for example, vegetable oil such as avocado oil, almond oil, oliveoil, sesame oil, safflower oil, soybean oil, camellia oil, apricotkernel oil, castor oil, and cotton seed oil; plant fats such as cacaobutter, coconut butter, palm oil, and Japan wax; and animal oils such asminke oil, egg yolk oil, and turtle oil.

[0048] The derivatives of cyclotetrasaccharide of the present inventionand the compositions comprising the same can be admixed with variouswaxes: for example, plant waxes such as jojoba oil, carnaba wax, andcandelila wax; animal-origin wax such as sperm whale oil, Baird's beakedwhale oil, beeswax, whale wax and lanoline; and mineral waxes such asmontan wax.

[0049] The derivatives of cyclotetrasaccharide of the present inventionand the compositions comprising the same can be admixed with varioushydrocarbons: for example, mineral hydrocarbons such as paraffin (alias“solid paraffin”), liquid paraffin, ceresin, microcryatalline wax, andvaseline; and hydrocarbons of animal origin such as squalane andsqualene.

[0050] The derivatives of cyclotetrasaccharide of the present inventionand the compositions comprising the same can be admixed with variousfatty acids: for example, lauric acid, myristic acid, palmitic acid,stearic acid, oleic acid, behenic acid, undecylic acid, lanoline fattyacid, hard lanoline fatty acid, soft lanoline fatty acid, isostearicacid, and those derivatives.

[0051] The derivatives of cyclotetrasaccharide of the present inventionand the compositions comprising the same can be admixed with variousalcohols: for example, higher alcohols (including polyhydric alcohols)such as lauryl alcohol, cethanol, cetostearyl alcohol, stearyl alcohol,oreyl alcohol, behenyl alcohol, lanoline alcohol, hydrogenated lanolinealcohol, hexyldecanol, octyldodecanol, and polyethyleneglycol; loweralcohols (including polyhydric alcohols) such as ethanol, propanol,isopropanol, butanol, ethyleneglycol, propyleneglycol, and glycerin; andtheir derivatives.

[0052] The derivatives of cyclotetrasaccharide of the present inventionand the compositions comprising the same can be admixed with variousesters: for example, hexyl laurate, isopropyl myristate, myristylmyristate, cetyl myristate, octyldodecyl myristate, isopropyl palmitate,butylstearate, cholesteryl stearate, chlesteryl acetate, cholesteryln-butyrate, cholesteryl caproate, cholesteryl laurate, cholesteryl12-hydroxystearate, decyl oreate, octyldodecyl oreate, lanolin-fattyacid isopropyl ester, glycerin trimyristate, propyleneglycol dioreate,myristyl lactate, cetyl lactate, lanorin acetate, and hexyldecyldimethyloctanoate; and their derivatives.

[0053] The derivatives of cyclotetrasaccharide of the present inventionand the compositions comprising the same can be admixed with varioussurfactants: for example, anionic surfactants such as zinc laurate, zincmyristiate, zinc palmitate, magnesium stearate, sodium laurylsulfate,triethanolamine laurylsulfate, sodium cetlysulfate, sodiumpolyoxyethylenelaurylether sulfate, triethanolaminepolyoxyethylenelaurylether sulfate, polyoxyethylenecetylether phosphate,polyoxyehylenealkylphenylether phosphate, sodium lauroylsarcosine,coconut oil fatty acid sarcosinetriethanolamine, sodiummethyltaurine-coconut oil fatty acid, and soybean phospholipid; cationicsurfactants such as stearyltrimethylammonium chloride,distearyldimethylammonium chloride, benzalconium chloride,cetylpyridinium chloride, alkylisoquinrinium bromide, anddodecyldimethyl-2-phenoxyethylammonium bromide; amphoionic surfactantssuch as sodium β-laurylaminopropionate, betainelauryldimethylaminoacetate, and betaine2-alkyl-N-carboxymethyl-N-hydroxyethylimidazorium; non-ionic surfactantssuch as self-emulsifying glycerin monostearate, lipophilic glycerinmonostearate, propyleneglycol dioreate, sorbitan monolauryate, sorbitanmonooreate, sucrose-fatty acid ester, monoethanolamide undecylenate,diethanolamide-coconut oil fatty acid ester, polyethyleneglycolmonooreate, myristyl lactate, polyoxyethylene-cetyl ether,polyoxyethylene-octylphenyl ether, polypxyethylenesorbit monolaurate,polyoxyethylenesorbitan monolaurate, polyoxyethylenesorbitanmonostearate, polyoxyethylenesorbitan trioreate, polyoxyethylenesorbittetraoreate, polyoxyethlene-castor oil and polyoxyethylene-hardeningcastor oil; and their derivatives.

[0054] The derivatives of cyclotetrasaccharide of the present inventionand the compositions comprising the same can be admixed with variouscoloring materials: for example, red-tar-colorings such as Amaranth,Erythrosine, Rose bengale, Acid red, Lake red C, Lithol red, Rhodamine,Brilliant Lake red, Eosin YS, Violamine R, Brilliant Fast scarlet, andPonceau R; orange-tar-colorings such as Dibromofluoresceine, Permanentorange, Erythrosine yellow NA, and Orange I; yellow-tar-colorings suchas Tartrazine, Sunset yellow, Uranine, Bentizine yellow G, Naphtholyellow S, and Yellow AB; green-tar-colorings such as Fast green FCF,Alizarin Cyanine green F, Light Green SF yellow, and Naphthol green B;blue-tar-colorings such as Brilliant blue FCF, Indigocarmine, Indigo,Patent blue NA, Carbanthrene blue, and Sudan blue; brown-tar-coloringssuch as Resorcin brown; purple-tar-colorings such as Alizurin purple andAlizurol purple; black-tar-colorings such as Naphthol blue black;inorganic colorings such as zinc oxide, titanium oxide, cobalthydroxide, aluminium hydroxide, talc, kaolin, mica, bentonite, manganeseviolet, and mica titanium; carotenoide-colorings such as β-carotene,lycopene, and crocin; flavonoide-colorings such as sisonine, saffloweryellow, rutin, quercetin, and hesperidin; flavin-colorings such asriboflavin; quinone-colorlings such as cochineal, alizarin and shikonin;and their derivatives.

[0055] The derivatives of cyclotetrasaccharide of the present inventionand the compositions comprising the same can be admixed with variousfragrant materials: for example, animal fragrant such as musk, civet,castoreum, and ambergris; plant fragrant such as distilled materials(essential oils) obtainable by steam distillation from fruit of anis,leaf of basil, fruit of caraway, cortex of cinnamon, seed of coriander,flower of lavender, seed of nutmeg, leaf of peppermint, flower of rose,seed or leaf of rosemary, and leaf of thymu; extracts (generallyclassified into absolutes, resinoids, oleoresins, and tinctures)obtainable from flower of hyacinth, flower of jasmine, flower of minosa,flower of rose, and seed of vanilla; synthetic fragrances such asacetophenone, anethole, benzylalcohol, butytlacetate, camphor, citral,citronellal, cuminaldehyde, estragole, ethylvanillin, geranylacetate,linalol, menthol, methyl-p-crezol, methyl salisylate, phenylacetic acid,and vanilline; and their derivatives. Further, prepared fragrancesproduced by suitably mixing those fragrances can be used in the presentinvention.

[0056] The derivatives of cyclotetrasaccharide of the present inventionand the compositions comprising the same can be admixed with varioushormones: for example, ovarian follicle hormones such as estrone andestradiol; corpus luteum hormones such as progesterone and pregnenolone;adrenal cortex hormones such as cortisone, hydrocortisone, prednisone,and prednisolone; and various vitamins: for example, compounds belongingvitamin A such as retinol, retinoic acid, α-, β-, and γ-carotene, andtheir derivatives; compounds belonging to vitamin B such as thiamine(vitamin B₁), riboflavin (vitamin B₂), pyridoxine, pyridoxal,pyridoxamine (hereinbefore, vitamin B₆), and their derivatives;compounds belonging to vitamin C such as L-ascorbic acid,glycosyl-L-ascorbic acids such as 2-O-α-D-glucosyl-L-ascorbic acid,acyl-derivatives of L-ascorbic acid and glycosyl-L-ascorbic acids (alias“lipophilic vitamin C”), other L-ascorbic acid derivatives such asL-ascorbic acid sulfate (sulfuric acid ester); compounds belonging tovitamin D such as ergocalciferol, cholecalciferol and their derivatives;and compounds belonging to vitamin E such as α-, β-, γ-, andδ-tocopherol, α-, β-, γ-, and δ-tocotrienol, and their derivatives.

[0057] In addition to the aforesaid plant extracts used as flavors, thederivatives of cyclotetrasaccharide of the present invention and thecompositions comprising the same can be admixed with various plantextracts: for example, mayweed extract, sage extract, aloe extract,salvia extract, Angelica keiskei extract, avocado extract, nettleextract, fennel extract, oolong tea extract, cork tree bark extract,barley extract, gumbo extract, orris extract, seaweed extract, Chinesequince extract, Glycyrrhiza glaba extract, quince seed extract, gardeniaextract, “kumazasa” extract, cinnamon extract, rice bran extract,fermented rice bran extract, stevia extract, celery extract, sialidextract, soybean extract, thymu extract, tea extract, camellia extract,Ligusticum acutilobum extract, corn extract, carrot extract, rugosa roseextract, cypress extract, loof ah extract, safflower extract, pineextract, peach extract, eucalyptus tree extract, strawberry geraniumextract, Chinese lemon extract, lily extract, adlay extract, tansyextract, Cyanobacteria extract, seaweed extract, apple extract,Memordica charantia extract, and lettuce extract; and compounds isolatedfrom plants such as hinokitiol, azulene, chlorophyll, and glycyrrhizin.A placenta extract can be used in the present invention as an animalextract.

[0058] The derivatives of cyclotetrasaccharide of the present inventionand the compositions comprising the same can be admixed with microbialextracts such as yeast extract. In addition to salts which are generallyaccepted and usually used, the derivatives of cyclotetrasaccharide ofthe present invention and the compositions comprising the same can beadvantageously admixed with natural salts (including solutions) such assea water, bittern, marine deep water, dried sea water, and mineralsalts.

[0059] The derivatives of cyclotetrasaccharide of the present inventionand the compositions comprising the same can be admixed with variousultraviolet-ray absorbing agent: for example, ethyl paraaminobenzoate,ethyl paraaminobenzoate hexyl ester, cinoxate, ethyl paradimethylcinnamate hexyl ester, 2-(2-hydroxy-5-methylphenyl)-benzotriazole,oxybenzozone, urocanynic acid, ethyl urocanyate, ruin, and quercetin;their derivatives; and organic compounds having an ultraviolet-rayshielding activity such as 5-chlorouracil, guanine, and cytosine; andphotosensitive dye: for example,2,2′[3′-[2-(3-heptyl-4-methyl-2-thiazoline-2-yliden)ethylidene]propenylene]-bis-[3-heptyl-4-methyl]thiazoliniumiodide (alias“PLATONIN”), 2-[2-(3-heptyl-4-methyl-2-thiazoline-2-ylidene)methyne]-3-heptyl-4-methylthiazoliniumiodide (alias “PIONIN”),6-[2-[(5-bromo-2-pyridyl)amino]vinyl]-1-ethyl-picoliniumiodide (alias“TAKANAL”), and 2-(2-anilinovinyl)-3,4-dimethyl-oxazoliniumiodide (alias“LUMINEX”); and their derivatives.

[0060] In addition to the aforesaid ingredients having an anti-oxidativeeffect, the derivatives of cyclotetrasaccharide of the present inventionand the compositions comprising the same can be admixed with variousanti-oxidizing agents: for example, propyl gallate, butyl gallate, actylgallate, dodecyl gallate, nordihydrogaialenic acid (alias “NDGA”),butylhydroxyanisole (alias “BHA”), dibutylhydroxytoluene (alias “BHT”),4-hydroxymethyl 1-2,6-ditertiarybutylphenol, catechin, rutin, andquercetin; and their derivatives.

[0061] In addition to the aforesaid ingredients having a preserving andsterilizing effect, the derivatives of cyclotetrasaccharide of thepresent invention and the compositions comprising the same can beadmixed with various preservatives and disinfectants: for example,phenolic compounds such as phenol, parachloromethacresol, resorcin,paraoxybenzoic acid ester, and cresol; acids (including their salts)such as benzoic acid, sorbic acid, salicylic acid, and boric acid;halogenized bisphenolic compounds such as hexachlorophen, bithionol, anddichlorophen; amides such as 3,4,4′-trichlorocarbanilide and undecylenicacid monoethanolamide; quaternal ammonium compounds such as benzalconiumchloride, benzetonium chloride, and decalinium chloride; compounds andtheir derivatives such as chlorohexidine hydrochloric acid,1-hydroxypyridine-2-thion, and lysozyme hydrochloric acid.

[0062] The derivatives of cyclotetrasaccharide of the present inventionand the compositions comprising the same can be admixed withantiperspirants and deodorants such as aluminum chloride, zinc chloride,chlorohydroxyl aluminum, allantoin chlorohydroxyl aluminum, and aluminumchlorohydrate; refrigerants such as menthol, spearmint oil, peppermintoil, camphor, thymol, spilanthol, and methylsalicylirate; and chelatingagents such as derivatives of ethylendiaminetetraacetate,tripolyphosphoric acid, hexamethacrylic acid, dihydroethylglycine,citric acid, tartaric acid, gluconic acid and sugar acid.

[0063] In addition to the aforesaid ingredients having an skin-whiteningeffect, the derivatives of cyclotetrasaccharide of the present inventionand the compositions comprising the same can be admixed with variousskin-whitening agents; for example, nucleic acid such as anti-senseoligonucleotides (for example, an anti-sense oligonucleotide againsttyrosinase gene); compounds such as kojic acid, lactic acid, anthranilicacid, coumarine, benzotriazole, imidazoline, pyrimidine, dioxane, furan,pyrone, nicotinic acid, albutin, baicalin, bicalen, and berberine; andtheir derivatives; inhibitor for melanine-formation, inhibitor fortyrosinase-formation, inhibitor of tyrosinase.

[0064] In addition to the aforesaid ingredients having ananti-inflammatory effect, the derivatives of cyclotetrasaccharide of thepresent invention and the compositions comprising the same can beadmixed with various anti-inflammatory agents; for example, allantoin,allantoin acetyl-D,L-methionine, allantoin β-glycyrrhetinoate,ichthammol, indometacin, acetylsalicylic acid, diphenhydramine chloride,guaiazulene, chamazulene, chlorpheniramine maleate, glycyrrhzinoic acid,glycyrrhetinoic acid, and oriental gromurell extract; and enzymes suchas protease, lipase, and lysozyme, which are originated frommicroorganisms such as Bacillus, Actinomycetes, and yeasts; plants; andanimals.

[0065] The derivatives of cyclotetrasaccharide of the present inventionand the compositions comprising the same can be admixed with varioussaccharides; for example, oligosaccharides such as xylose, arabinose,glucose, fructose, galactose, tagatose, sucrose, maltose, lactose,trehalose, panose, maltotriose, and maltotetraose; cyclic saccharidessuch as cyclotetrasaccharide, and α-, β-, and γ-cyclodextrin; sugaralcohols such as xylitol, arabitol, sorbitol, maltitol, and mannitol;polysaccharide, their derivatives and partial hydrolyzates such ashyaluronic acid, chondroitin sulfate, pullulan, cellulose, starch,dextran, pectin, carrageenan, locust bean gum, guar gum, corn syrup, gumarabic, tragacanth gum, xanthan gum, glucomannan, and chitin.

[0066] The derivatives of cyclotetrasaccharide of the present inventionand the compositions comprising the same can be admixed with variousamino acids; for example, glycine, serine, threonine, tyrosine,cysteine, cystine, asparagine, glutamine, pyrolidone carboxylic acid,hydroxyproline, pipecolic acid, sarcosine, homocysteine, homoserine,citrulline, aspartic acid, glutamic acid, cysteine sulfinic acid,algininosuccinic acid, arginine, lysine, histidine, ornithine, alanine,valine, leucine, isoleucine, methionine, phenylalanine, tryptophan,proline, β-alanine, taurine, β-aminobutylic acid, and γ-aminobutylicacid; and their salts.

[0067] In addition to the aforesaid ingredients having a thickeningeffect, the derivatives of cyclotetrasaccharide of the present inventionand the compositions comprising the same can be admixed withwater-soluble polymers; for example, quince seed, sodium alginate,cationated cellulose, hydroxyethylcellulose, carboxymethylcellulose,carboxymethylstarch, propylenglycol alginate, collagen, keratin, casein,albumin, gelatin, hydroxypropil-trimethylammoniumchloride-ether,polyvinylalcohol, polyvinylpyrolidone, polivinylpyrolidone-vinylacetatecopolymerizate, polyethyleneimine, sodium polyacrylate,polyvinylmethylether, and carboxyvinylpolymer; electolyte such as sodiumchloride, potassium chloride, and sodium sulfate; and various oils.

[0068] Whole examples of ingredients, which are able to form salt, arenot described in the above explanation. However, salts acceptable to theobject can be arbitrarily used in the present invention even though theyare not described.

[0069] The methods for incorporating the aforesaid various ingredientsinto derivatives of cyclotetrasaccharide or the composition comprisingthe same are those which can incorporate such ingredients into a productbefore completion of their processing, and which can be appropriatelyselected from the following conventional methods; mixing, kneading,dissolving, melting, soaking, penetrating, dispersing, applying,coating, spraying, injecting, crystallizing, and solidifying. It ispreferable to incorporate such ingredients in an amount of, usually,0.1% or higher, desirably, 1% or higher.

[0070] Derivatives of cyclotetrasaccharide of the present invention andthe compositions comprising the same can be shaped intact or, ifnecessary, after mixing with fillers, excipients, and binders, intovarious shapes such as granules, spheres, sticks, plates, cubes, andtablets.

[0071] Varying depend on an introduced substituent, derivatives ofcyclotetrasaccharide of the present invention and the compositionscomprising the same show sweetness in the same or adjusted level withcyclotetrasaccharide, well harmonize with other tastable materialshaving sour-, salty-, bitter-, astringent-, delicious-, andbitter-taste, and have a high acid- and heat-tolerance. Thus, they canbe advantageously used to sweeten, improve tastes and qualities ofgeneral foods and beverages. Derivatives of cyclotetrasaccharide or thecompositions comprising the same can be advantageously used as asweetener, taste-improving agent, quality-improving agent for variousfoods: for example, soy sauce, powdered soy sauce, miso, “funmatsu-miso”(a powdered miso), “moromi” (a refined sake), “hishio” (a refined soysauce), “furikake” (a seasoned fish meal), mayonnaise, dressing,vinegar, “sanbai-zu” (a sauce of sugar, soy sauce and vinegar),“funmatsu-sushi-zu” (powdered vinegar for sushi), “chuka-no-moto” (aninstant mix for Chinese dish), “tentsuyu” (a sauce for Japanese deep fatfried food), “mentsuyu” (a sauce for Japanese vermicelli), sauce,catsup, “yakiniku-no-tare” (a sauce for Japanese grilled meat), curryroux, instant stew mix, instant soup mix, “dashi-no-moto” (an instantstock mix), mixed seasoning, “mirin” (a sweet sake), “shin-mirin” (asynthetic mirin), table sugar, and coffee sugar. Also, derivatives ofcyclotetrasaccharide and the compositions comprising the same can beadvantageously used to sweeten and improve the taste and quality of“wagashi” (Japanese cakes) such as “senbei ” (a rice cracker), “arare”(a rice cake cube), “okoshi” (a millet and rice cake), “gyuhi” (a starchpaste), “mochi” (a rise paste) and the like, “manju” (a bun with abean-jam), “uiro” (a sweet rice jelly), “an” (a bean-jam) and the like,“yokan” (a sweet jelly of beans), “mizu-yokan” (a soft azuki-beanjelly), “kingyoku” (a kind of yokan), jelly, pao de Castella, and“amedama” (a Japanese toffee); western confectioneries such as bun,biscuit, cracker, cookie, pie, pudding, butter cream, custard cream,cream puff, waffle, sponge cake, doughnut, chocolate, chewing gum,caramel, nougat, and candy; frozen desserts such as ice cream andsherbet; syrups such as a “kajitsu-no-syrup-zuke” (a preserved fruit)and “korimitsu” (a sugar syrup for shaved ice); pastes such as flourpaste, peanut paste, and fruit paste; processed fruits and vegetablessuch as jam, marmalade, “syrup-zuke” (fruit pickles), and “toka”(conserves); pickles and pickled products such as a “rukujin-zuke” (redcolored radish pickles), “bettara-zuke” (a kind of whole fresh radishpickles), “senmai-zuke” (a kind of sliced fresh radish pickles), and“rakkyo-zuke” (pickled shallots); premix for pickles and pickledproducts such as “takuan-zuke-no-moto” (a premix for pickled radish),and “hakusai-zuke-no-moto” (a premix for fresh white rape pickles); meatproducts such as ham and sausage; products of fish meat such as a fishham, fish sausage, “kamaboko” (a steamed fish paste), “chikuwa” (a kindof fish paste), and “tenpura” (a Japanese deep-fat fried fish paste);“chinmi” (relish) such as a “uni-no-shiokara” (salted guts of urchin),“ika-no-shiokara” (salted guts of squid), “su-konbu” (processed tangle),“saki-surume” (dried squid strips), “fugu-no-mirin-boshi” (a driedmirin-seasoned swellfish), seasoned fish flour such as of Pacific cod,sea bream, shrimp, etc.; “tsukudani” (foods boiled down in soy sauce)such as those of laver, edible wild plants, dried squid, small fish, andshellfish; daily dishes such as a “nimame” (cooked beans), potato salad,and “konbu-maki” (a tangle roll); milk products; canned and bottledproducts such as those of meat, fish meat, fruit, and vegetable;alcoholic beverages such as synthetic sake, fermented liquor, sake,fruit liquor, low-malt beer, and beer; soft drinks such as coffee,cocoa, juice, carbonated beverage, sour milk beverage, and beveragecontaining a lactic acid bacterium; instant food products such asinstant pudding mix, instant hot cake mix, instant juice, instantcoffee, “sokuseki-shiruko” (an instant mix of azuki-bean soup with ricecake), and instant soup mix; and other foods and beverages such as solidfoods for babies, foods for therapy, drinks, peptide foods, and frozenfoods. Derivatives of cyclotetrasaccharide or the compositionscomprising the same can be also used for improving preference andphysical properties of feeds and pet foods for animals and pets such asdomestic animals, poultry, honeybees, silk warms, and fishes. Inaddition, derivatives of cyclotetrasaccharide or the compositionscomprising the same can be advantageously used as preference-improvingagent, taste-improving agent, masking agent, quality-improving agent andstabilizer for various compositions such as favorite foods, cosmetics,and pharmaceuticals in a solid, paste, or liquid form such as tobacco,cigarette, tooth paste, lipstick, rouge, lip cream, internal liquidmedicine, tablet, troche, cod-liver oil in the form of drop, oralrefrigerant, cachou, and gargle. When used as a quality-improving agentor stabilizer, a derivative of cyclotetrasaccharide and the compositionscomprising the same can be advantageously incorporated into biologicallyactive substances susceptible to lose their effective ingredients andactivities, as well as into health foods and pharmaceuticals containingthe biologically active substances. Health foods and pharmaceuticals,having a satisfactory stability and quality, in a liquid, paste, orsolid form can be easily produced without losing effective ingredientsand activities of biologically active substances: for example, liquidpreparations containing lymphokines such as α-, β-, and γ-interferons,tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β),macropharge migration inhibitory factor, colony-stimulating factor,transfer factor, and interleukin II; liquid preparations containinghormones such as insulin, growth hormone, prolactin, erythropoietin, andfollicle-stimulating hormone; liquid preparations containing biologicalpreparations such as BCG vaccine, Japanese encephalitis vaccine, measlesvaccine, live polio vaccine, small pox vaccine, tetanus toxin,Trimeresurus antitoxin, and human immunoglobulin; liquid preparationscontaining antibiotics such as penicillin, erythromycin,chloramphenicol, tetracycline, streptmycin, and kanamycin sulfate;liquid preparations containing vitamins such as thiamin, riboflavin,L-ascorbic acid, cod liver oil, carotenoide, ergosterol, and tocopherol;highly unsaturated fatty acids or their ester derivatives such as EPA,DHA, and arachidonic acid; liquid preparations containing enzymes suchas lipase, esterase, urokinase, protease, β-amylase, isoamylase,glucanase, and lactase; extracts such as ginseng extract, turtleextract, chlorella extract, aloe extract, and propolis extract;biologically active substances such as royal jelly; livingmicroorganisms pastes of virus, lactic acid bacteria, and yeast.

[0072] The following explains a process for producing enzymes which arenecessary for producing a material of the present invention,cyclotetrasaccharide.

EXAMPLE A-1

[0073] Preparation of Enzymes for Producing Cyclotetrasaccharide:Cultivation of Bacillus globisporous C9

[0074] α-Isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme, which are used for producingcyclotetrasaccharide using starch as material, were prepared. A liquidculture medium consisting 4% (w/v) of “PINE-DEX #4”, a partial starchhydrolyzate commercialized by Matsutani Chemical Industry Co., Ltd.,Hyogo, Japan, 1.8% (w/v) of “ASAHIMEAST”, ayeast extract commercializedby Asahi Brewery Ltd., Tokyo, Japan, 0.1% (w/v) of dipotassiumphosphate, 0.06% (w/v) of sodium phosphate dodeca-hydrate, 0.05% (w/v)of magnesium sulf ate heptahydrate, and water was placed in 500-mlErlenmeyer flasks in a respective amount of 100 ml, sterilized byautoclaving at 121° C. for 20 min, cooled and seeded with Bacillusglobisporus C9, FERM BP-7143, followed by culturing under rotary-shakingconditions at 27° C. and 230 rpm for 48 hours for a seed culture. About20 L of a fresh preparation of the same liquid culture medium as used inthe above seed culture were placed in a 30 L fermentor, sterilized byheating, and then cooled to 27° C., and inoculated with 1% (v/v) of theseed culture, followed by culturing at 27° C. and pH 6.0 to 8.0 for 48hours under aeration-agitation conditions. About 18 L of supernatant,obtained by centrifuging at 10,000 rpm for 30 min, had about 0.45units/ml of α-isomaltosylglucosaccharide-forming enzyme activity, i.e.,a total activity of about 8,110 units; 1.5 unit ofα-isomaltosyl-transferring enzyme activity, i.e., a total enzymaticactivity of about 26,900 units; and 0.95 unit/ml ofcyclotetrasaccharide-forming activity, i.e., a total activity of about17,100 units. The activities of enzymes were measured as follows:

[0075] The activity of α-isomaltosylglucosaccharide-forming enzyme wasmeasured by the following assay: A substrate solution was prepared bydissolving maltotriose in 100 mM acetate buffer (pH 6.0) to give aconcentration of 2% (w/v). A reaction mixture was prepared by mixing 0.5ml of the substrate solution and 0.5 ml of an enzyme solution, andincubated at 35° C. for 60 min. After stopping the reaction by boilingfor 10 min, the amount of maltose formed with isomaltosylmaltose in thereaction mixture was determined by high-performance liquidchromatography (HPLC) according to the conventional method. One unit ofα-isomaltosylglucosaccharide-forming activity was defined as the amountof the enzyme that forms one μmole of maltose per minute under the aboveconditions. The activity of α-isomaltosylglucosaccharide-forming enzymeis represented by the unit measured by the above assay throughout thespecification.

[0076] The activity of α-isomaltosyl-transferring enzyme was measured bythe following assay: A substrate solution was prepared by dissolvingpanose in 100 mM acetate buffer (pH 6.0) to give a concentration of 2%(w/v). A reaction mixture was prepared by mixing 0.5 ml of the substratesolution and 0.5 ml of an enzyme solution, and incubated at 35° C. for30 min. After stopping the reaction by boiling for 10 min, the amount ofglucose formed with cyclotetrasaccharide in the reaction mixture wasdetermined by the glucose oxidase-peroxidase method. One unit ofα-isomaltosyl-transferring enzyme activity was defined as the amount ofthe enzyme that forms one μmole of glucose per minute under the aboveconditions. The activity of α-isomaltosyl-transferring enzyme isrepresented by the unit measured by the above assay throughout thespecification.

[0077] Cyclotetrasaccharide-forming activity was measured by thefollowing assay: A substrate solution was prepared by dissolving“PINE-DEX #100”, a partial starch hydrolyzate commercialized byMatsutani Chemical Industry Co., Ltd., Hyogo, Japan, in 50 mM acetatebuffer (pH 6.0) to give a concentration of 2% (w/v). A reaction mixturewas prepared by mixing 0.5 ml of the substrate solution and 0.5 ml of anenzyme solution, and incubated at 35° C. for 60 min. After stopping thereaction by boiling for 10 min, one ml of 50 mM acetate buffer (pH 5.0)containing 70 units/ml of “TRANSGLUCOSIDASE L AMANO™”, an α-glucosidaseproduced by Amano Enzyme Inc., Nagoya, Japan, and 27 units/ml ofglucoamylase, commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan,was added to the reaction mixture and the resultant mixture wasincubated at 50° C. for 60 min. After stopping the reaction by heatingat 100° C. for 10 min, the amount of cyclotetrasaccharide was determinedby a conventional HPLC method. One unit of cyclotetrasaccharide-formingactivity was defined as the amount of enzyme that that forms one μmoleof glucose per minute under the above conditions.

EXAMPLE A-2

[0078] Preparation of Enzymes Originated from Bacillus globosporus C9

[0079] About 18 L of the culture supernatant obtained in Example A-1were salted out with 80% saturated ammonium sulfate solution and allowedto stand at 40° C. for 24 hours, and the formed precipitates werecollected by centrifuging at 10,000 rpm for 30 min, dissolved in 10 mMsodium phosphate buffer (pH 7.5), and dialyzed against the same bufferto obtain about 400 ml of a crude enzyme solution. The crude enzymesolution had 8,110 units of α-isomaltosylglucosaccharide-forming enzyme,24,700 units of α-isomaltosyl-transferring enzyme, and about 15,600units of cyclotetrasaccharide-forming activity. The crude enzymesolution was subjected to ion-exchange column chromatography using 1,000ml of “SEPABEADS FP-DA13” gel, an ion-exchange resin commercialized byMitsubishi Chemical Industries, Ltd., Tokyo, Japan. Bothα-isomaltosylglucosaccharide-forming enzyme andα-isomaltosyl-transferring enzyme and were eluted as non-adsorbedfractions without adsorbing on “SEPABEADS FP-DA13” gel. The fractionswere collected and dialyzed against 10 mM sodium phosphate buffer (pH7.0) with 1 M ammonium sulfate. The dialyzed solution was centrifuged toremove impurities, and subjected to affinity column chromatography using500 ml of “SEPHACRYL HR S-200” gel, a gel commercialized by AmershamBioscience K. K., Tokyo, Japan. Both enzymes adsorbed on “SEPHACRY HRS-200” gel and, when sequentially eluted with a linear gradientdecreasing from 1 M to 0 M of ammonium sulfate and a linear gradientincreasing from 0 mM to 100 mM of maltotetraose,α-isomaltosylglucosaccharide-forming enzyme and theα-isomaltosyl-transferring enzyme were separately eluted, i.e., theformer was eluted with a linear gradient of maltotetraose at about 30 mMand the latter was eluted with a linear gradient of ammonium sulfate atabout 0 M. Thus, fractions with the α-isomaltosylglucosaccharide-forming enzyme activity and those with the α-isomaltosyl-transferringenzyme activity were separately collected.

[0080] The followings describe the methods for purifyingα-isomaltosylglucosaccharide-forming enzyme and α-isomaltosyltransferring enzyme, respectively.

EXAMPLE A-3

[0081] Purification of α-isomaltosylglucosaccharide-forming EnzymeOriginated from Bacillus globisporus C9

[0082] The fractions comprising α-isomaltosylglucosaccharide-formingenzyme, obtained in Example A-2 was dialyzed against10 mM sodiumphosphate buffer (pH 7.0) with 1 M ammonium sulfate. The dialyzedsolution was centrifuged to remove impurities, and subjected tohydrophobic column chromatography using 350 ml of “BUTYL-TOYOPEARL 650M”gel, a hydrophobic gel commercialized by Tosoh Corporation, Tokyo,Japan. The enzyme adsorbed on “BUTYL-TOYOPEARL 650M” gel and, wheneluted with a linear gradient decreasing from 1 M to 0M of ammoniumsulfate, the enzyme was eluted at an ammonium sulfate concentration ofabout 0.3 M, and fractions with the enzyme activity were collected. Thecollected solution was dialyzed against 10 mM sodium phosphate buffer(pH 7.0) with 1 M ammonium sulfate again. The dialyzed solution wascentrifuged to remove impurities, and purified by affinitychromatography using “SEPHACRYL HR S-200” gel.

EXAMPLE A-4

[0083] Purification of α-isomaltosyl-transferring Enzyme Originated fromBacillus globisporus C9

[0084] The fractions comprising α-isomaltosyl-transferring enzyme, whichare separated from those comprising α-isomaltosylgucosaccharide-formingenzyme by the affinity chromatography described in Example A-2, wasdialyzed against 10 mM sodium phosphate buffer (pH 7.0) with 1 Mammonium sulfate. The dialyzed solution was centrifuged to removeimpurities, and subjected to hydrophobic column chromatography using 350ml of “BUTYL-TOYOPEARL 650M” gel, a hydrophobic gel commercialized byTosoh Corporation, Tokyo, Japan. The enzyme adsorbed on “BUTYL-TOYOPEARL650M” gel and, when eluted with a linear gradient decreasing from 1 M to0M of ammonium sulfate, the enzyme was eluted at an ammonium sulfateconcentration of about 0.3 M, and fractions with the enzyme activity wascollected. The collected solution was dialyzed against 10 mM sodiumphosphate buffer (pH 7.0) with 1 M ammonium sulfate again. The dialyzedsolution was centrifuged to remove impurities, and purified by affinitychromatography using “SEPHACRYL HR S-200” gel.

[0085] The following describes a process for producingcyclotetrasaccharide as material of the present invention.

EXAMPLE B-1

[0086] Preparation of Cyclotetrasaccharide

[0087] A corn phytoglycogen, commercialized by Q.P. Corporation, Tokyo,Japan, was prepared into a 15% (w/v) solution, adjusted to pH 6.0 and at30° C.; admixed with 1 unit/g-dry solid of purifiedα-isomaltosylglucosaccharide-forming enzyme, obtained in Example A-3,and 10 units/g-dry solid of purified α-isomaltosyl-transferring enzyme,obtained in Example A-4; incubated for 48 hours; and then heated at 100°C. for 10 min to inactivate enzymes. After adjusting the reactionmixture at pH 5.0 and 45° C., the remaining reducing oligosaccharideswere hydrolyzed by α-glucosidase and glucoamylase as in the case ofExample A-1. Then, the resulting reaction mixture was adjusted at pH 5.8with sodium hydroxide and kept at 90° C. for one hour to inactivateenzymes. The resulting insoluble material was removed by filtration.After concentrating the resulting filtrate using a reverse osmosismembrane to give a concentration of about 16%, on a dry solid base,about 6.2 kg of a saccharide solution containing about 3,700 g of drysolid was obtained by decolorizing, deionizing, filtrating, andconcentrating the solution according to the conventional manner.

[0088] The resulting saccharide solution was subjected to a columnchromatography using about 225 liters of “AMBERLITE CR-1310 (Na-form)”,an ion-exchanger resin commercialized by Japan Organo Co., Ltd., Tokyo,Japan, and fractionated under the conditions at a column temperature of60° C. and a flow rate of about 45 L/hour. The fractions, showingcyclotetrasaccharide purity of 98% or higher by monitoring thesaccharide composition of eluate by HPLC, were collected.

EXAMPLE B-2

[0089] Preparation of Hydrous Crystalline Cyclotetrasaccharide

[0090] The fractions containing cyclotetrasaccharide having a purity ofabout 98% or higher, which were obtained by the above method, were mixedand the resulting solution was concentrated to give a concentration ofabout 50%, on a dry solid basis, using a evaporator. Then, about fivekilograms of the concentrated solution was placed in a cylindricalcontainer and crystallized by cooling the temperature from 65° C. to 20°C. with taking a time of 20 hours, and white crystalline powder wasobtained. Successively, 1,360 grams wet-weight of the crystallinematerial was collected by separating the massecuites with a centrifugalfiltration apparatus. Further, 1,170 grams of crystallinecyclotetrasaccharide powder was obtained by drying the above crystallinematerial at 60° C. for three hours. The product showed an extremely highcyclotetrasaccharide purity of 99.9% or higher when sugar composition ofthe product was analyzed by HPLC. The crystalline powder showed adiffraction spectrum having major diffraction angles (2θ) of 10.1°,15.2°, 20.3°, and 25.5° by X-ray diffraction analysis. Also, since thecrystalline powder was found to have a moisture content of about 13.0%by the Karl-Fischer method, it was revealed that the crystalline powderwas a crystal comprising 5-6 molecules of water per one molecule ofcyclotetrasaccharide.

EXAMPLE B-3

[0091] Preparation of Anhydrous Crystalline Cyclotetrasaccharide

[0092] Crystalline cyclotetrasaccharide powder, obtained by the methodof Example B-2, was dried in vacuo at 120° C. for 16 hours. Theresulting crystalline powder showed a diffraction spectrum having majordiffraction angles (2θ) of 10.8°, 14.7°, 15.0°, and 21.5° by X-raydiffraction analysis. Also, since the crystalline powder was found tohave a moisture content of about 0.2% by the Karl-Fischer method, it wasrevealed that the crystalline powder was substantially in anhydrousform.

EXAMPLE B-4

[0093] Preparation of Anhydrous Cyclotetrasaccharide in Amorphous Form

[0094] The fractions containing cyclotetrasaccharide having a purity ofabout 98% or higher, which were obtained by the method of Example B-1,were mixed and the resulting solution was concentrated to give aconcentration of 50%. After freezing the concentrated solution rapidlyto −80° C., the resulting frozen material was dried by freeze-drying andfurther dried in vacuo at 80° C. for three hours, and the resultingdried material was pulverized using a pulverizer. The powder showed adiffraction spectrum having no peaks by X-ray diffraction analysis,suggesting the absence of crystal. Therefore, it was revealed that thepowder was in amorphous form. Further, since the powder was found tohave a moisture content of about 0.3% by the Karl-Fischer method, it wasrevealed that the powder was substantially in anhydrous form.

[0095] Cyclotetrasaccharide can be produced from starch by enzymaticreaction using enzymes, which are originated from microorganisms otherthan Bacillus globisporus C9 such as Bacillus globisporus C11 and N75,Arthrobacter ramosus S1, and Arthrbacter globiformis A19, as well as theprocedures described above.

[0096] The following describes a process for producing the derivative ofcyclotetrasaccharide of the present invention.

EXAMPLE C-1

[0097] Benzyl Derivative of Cyclotetrasaccharide

[0098] Five parts by weight of anhydrous crystallinecyclotetrasaccharide, obtained by the method of Example B-3, and 37parts by weight of potassium hydroxide were dissolved in 64 parts byweight of benzyl chloride, and then the resulting solution was heated at140° C. for three hours. After cooling the solution to the ambienttemperature, 200 parts by weight of distilled water and 400 parts byweight of ethyl acetate were added to the solution and mixed. Afterstanding the solution to separate aqueous phase and ethyl acetate phase,the resulting ethyl acetate phase was collected. After dehydrating thesolution with suitable amount of anhydrous magnesium sulfate accordingto the conventional method, the solution was dried under a reducedpressure to obtain benzyl cyclotetrasaccharide.

[0099] It was revealed that the product had benzyl groups with anaverage degree of substitution of 7.3 by measuring the degree ofsubstitution using a spectrophotometric analysis measuring theadsorption at 262 nm of benzene ring according to the conventionalmethod. Since the product is a lipophilic substance, it can beadvantageously incorporated into conventional oily cosmetics.

EXAMPLE C-2

[0100] Methyl Derivative of Cyclotetrasaccharide

[0101] Five parts by weight of anhydrous amorphous cyclotetrasaccharide,obtained by the method of Example B-4, was dissolved in 125 parts byweight of anhydrous dimethylsulfoxide, and then 12.5 parts by weight ofsodium hydride was added to the solution and mixed. After cooling thesolution for 10 min in an ice bath, it was heated at 60° C. for twohours. Successively, 22.5 parts by weight of methyl iodide was graduallyadded to the solution with cooling in an ice bath, and the resultingsolution was stirred at the ambient temperature for 18 hours. Afteradding 40 parts by weight of methanol to the resulting solution, 200parts by weight of distilled water was further added to the mixture.After adding 500 parts by weight of chloroform to the resulting solutionand stirring, the mixture was stand to separate aqueous phase andchloroform phase, and then the chloroform phase was collected. Afteradding 50 parts by weight of distilled water to the chloroform solution,stirring, and standing, the resulting chloroform phase was collectedagain. After repeating the procedure ten times, the resulting solutionwas dehydrated with suitable amounts of anhydrous magnesium sulfate.After concentrating the solution, 100 parts by weight of saturatedsodium chloride solution was added to the concentrate and the resultingsolution was stirred at 60° C. for 30 min. After cooling the solution inan ice bath, the resulting supernatant (aqueous phase) was removed. Theabove procedure was repeated once again. The resultant was dissolved in300 parts by weight of chloroform, stirred at 60° C. for 30 min,dehydrated with suitable amounts of anhydrous magnesium sulfate, andconcentrated to obtain syrupy methyl cyclotetrasaccharide.

[0102] It was revealed that the product had methyl groups with anaverage degree of substitution of 7.5 by ¹H-NMR analysis. Since theproduct is a lipophilic substance, it can be advantageously incorporatedinto conventional oily cosmetics.

EXAMPLE C-3

[0103] Linoleic Acid Ester of Cyclotetrasaccharide

[0104] Ten parts by weight of anhydrous crystallinecyclotetrasaccharide, obtained by the method of Example B-3, and 200parts by weight of anhydrous pyridine were placed in a reaction vessel,and then four parts by weight of thiazolithion-linoleic acid amide,which is dissolved in five parts by weight of anhydrous pyridine, wasfurther added to the solution under the ventilation of argon. Themixture was further admixed with 0.085 part by weight of 60% (w/w) oilysodium hydride and reacted at ambient temperature for two hours. Afteradding 1:5 parts by weight of saturated aqueous ammonium chloridesolution, pyridine was removed under a reduced pressure, and 11.2 partsby weight of the residue was obtained. Then, the residue was purifiedusing silica gel chromatography to obtain linolenic acid ester ofcyclotetrasaccharide.

[0105] The product, having no taste or scent and a high activity, can beadvantageously incorporated as a non-ionic surfactant into foods,cosmetics, and pharmaceuticals.

EXAMPLE C-4

[0106] Myristic Acid Ester of Cyclotetrasaccharide

[0107] Two hundred parts by weight of anhydrous amorphouscyclotetrasaccharide, obtained by the method of Example B-4, wasdissolved in 800 parts by weight of N,N′-dimethylformamide, and 600parts by weight of myristic acid methyl ester and four parts by weightof calcium carbonate were further added to the solution. The mixture wasreacted with stirring at 85-95° C. for 24 hours under the reducedpressure of 100-200 mmHg. Successively, the reactant was evaporated toremove solvent, and the resulting residue was extracted two times using300 parts by weight of acetone per once. After concentrating the extractand washing with benzene and ether, the resulting gummy oily substancewas soaked into 300 parts by weight of acetone and extracted. Theextract was cooled in an ice bath, and the resulting precipitate wascollected and dried to obtain myristic acid ester ofcyclotetrasaccharide.

[0108] The product, having no taste or scent and a high activity, can beadvantageously incorporated as a non-ionic surfactant into foods,cosmetics, and pharmaceuticals.

EXAMPLE C-5

[0109] Dodecyl Ether of Cyclotetrasaccharide

[0110] After placing 390 parts by weight of n-dodecanol into a reactionvessel and heating to 125° C., one part by weight of p-toluene sulfonicacid as a catalyst was added to the solution, and then the pressure inthe vessel was reduced to 5-10 mmHg. Separately, 100 parts by weight ofanhydrous crystalline cyclotetrasaccharide, obtained by the method inExample B-3, was suspended in 130 parts by weight of n-dodecanol. Thesuspension was added into the above vessel drop by drop in a flow rateof 2.3 parts by weight/min with taking a time of 100 min for thereaction. Successively, the reactant was neutralized with aqueous sodiumcarbonate solution, and cyclotetrasaccharide dodecyl ether was obtainedby evaporating residual alcohol.

[0111] The product having a high activity can be advantageouslyincorporated as a surfactant into conventional detergents such asdetergent for washing, for kitchen, shampoo, and etc.

EXAMPLE C-6

[0112] Sulfate Ester of Cyclotetrasaccharide

[0113] After placing one part by weight of anhydrous crystallinecyclotetrasaccharide, obtained by the method of Example B-3, five partsby weight of sulfuric anhydride-dimethylformamide complex, separatelyprepared according to the conventional method, was added to thesaccharide drop by drop and reacted at the ambient temperature for fourhours and then further reacted at 70° C. for one hour. Afterneutralizing by adding suitable amount of 5 N sodium hydroxide solution,five-folds in volume of methanol was added to the solution and stood fora while. The resulting precipitate was collected by filtrating under areduced pressure to obtain sulfate ester of cyclotetrasaccharide.

[0114] The product having a high quality can be advantageouslyincorporated into conventional cosmetics as a moisture-retaining agentand skin-care agent.

EXAMPLE C-7

[0115] Sulfate Ester of Cyclotetrasaccharide

[0116] One hundred parts by weight of anhydrous amorphouscyclotetrasaccharide, obtained by the method in Example B-4, wassulfated by the method of Example C-6, and a composition comprisingsulfate ester of cyclotetrasaccharide was obtained.

[0117] The product having a high quality can be advantageouslyincorporated into conventional cosmetics as a moisture-retaining agentand skin-care agent.

EXAMPLE C-8

[0118] Cyanuric Acid Derivative of Cyclotetrasaccharide

[0119] Two parts by weight of anhydrous crystallinecyclotetrasaccharide, obtained by the method in Example B-3, wassuspended in 20 parts by weight of N,N′-dimethylformamide, comprising acatalyst amount of pyridine and 5% (w/v) of cyanuric chloride, andreacted at the ambient temperature for three hours. The reaction mixturewas filtrated, and the resulting residue was washed with acetone anddried to obtain cyanuric acid derivative of cycoltetrasaccharide.

[0120] The product can be bound with organic compounds such as proteinsand nucleic acids.

EXAMPLE C-9

[0121] Tosyl-derivative of Cyclotetrasaccharide

[0122] Fifteen parts by weight of anhydrous crystallinecyclotetrasaccharide, obtained by the method in Example B-3, wassuspended in 50 parts by weight of pyridine. Twelve parts by weight ofp-toluene-sulfonylchloride was added to the suspension and stirred at 0°C. for 18 hours. The reactant was extracted with ethyl acetate, and theresulting extract was washed with diluted hydrochloric acid and brine.The washed extract was concentrated by drying to obtain tosyl-derivativeof cyclotetrasaccharide.

[0123] The product is useful as an intermediate for various derivatives.

EXAMPLE C-10

[0124] Phenylsulfide Derivative of Cyclotetrasaccharide

[0125] Seven parts by weight of potassium t-butoxide was dissolved in 20parts by weight of dimethylformamide, and seven parts by weight ofthiophenol was further added drop by drop to the resulting solutionunder cooling to 0° C. and stirred at 0° C. for 10 min. Fourteen partsby weight of tosyl-derivative, prepared in Example C-9, was dissolved indimethylformamide, added to the above solution, and stirred at theambient temperature for one hour. The solution was extracted with ethylacetate, and the resulting extract was washed with brine. Then, thewashed extract was concentrated. The resulting residue (concentrate) waspurified by silica gel chromatography to obtain phenylsulfide derivativeof cyclotetrasaccharide.

[0126] The product is a lipophilic substance and can be used tocosmetics and pharmaceuticals.

EXAMPLE C-11

[0127] Amino Derivative of Cyclotetrasaccharide

[0128] Ten parts by weight of tosyl-derivative of cyclotetrasaccharide,prepared in Example C-9, was dissolved in 20 parts by weight ofanhydrous dimethylformamide in the presence of nitrogen gas. One part byweight of anhydrous sodium azide was further added to the solution, andthe resulting mixture was stirred at 65° C. for 18 hours in the presenceof nitrogen gas. After cooling the solution to the ambient temperature,150 parts by weight of water cooled with ice was added to the solution.The resulting precipitate was collected and dried to obtaindiazo-derivative. Successively, one part by weight of thediazo-derivative was dissolved in the mixture of 100 parts by weight ofpurified dioxane and 20 parts by weight of distilled methanol. Fourparts by weight of purified triphenylphosphine was added to the solutionwhile stirring in the presence of nitrogen gas and further stirred forone hour. Then, five parts by weight of concentrated aqueous ammonia wasadded drop by drop to the above solution and the resultant was stirredfor 12 hours in the presence of nitrogen gas. After evaporating thesolvent, the product was suspended in 250 parts by weight of water, andthe pH of the solution was set to pH 4 with 1 N hydrochloric acid. Thesuspension was washed three-times with 500 parts by weight of benzene toremove triphenylphosphineoxide. The resultant was freeze-dried to obtainamino derivative of cyclotetrasaccharide.

[0129] The product has an ability to bind with organic compounds havingcarboxyl groups and can be useful as an intermediate for introducingother substituents.

[0130] The present inventors deposited Bacillus globisporus C9 (FERMBP-7143), described in the present specification, on Apr. 25, 2000, inInternational Patent Organism Depositary National Institute of AdvancedIndustrial Science and Technology, Tsukuba Central 6, 1-1, Higashi1-Chome Tsukuba-shi, Ibaraki-ken, 305-8566, Japan. The present inventorsalso deposited Bacillus globisporus C11 (FERM BP-7144), described in thepresent specification, on Apr. 25, 2000, in International PatentOrganism Depositary National Institute of Advanced Industrial Scienceand Technology, Tsukuba Central 6, 1-1, Higashi 1-Chome Tsukuba-shi,Ibaraki-ken, 305-8566, Japan. The present inventors also depositedBacillus globisporus N75 (FERM BP-7591), described in the presentspecification, on May 16, 2001, in International Patent OrganismDepositary National Institute of Advanced Industrial Science andTechnology, Tsukuba Central 6, 1-1, Higashi 1-Chome Tsukuba-shi,Ibaraki-ken, 305-8566, Japan. The present inventors also depositedArthrobacter globiformis A19 (FERM BP-7590), described in the presentspecification, on May 16, 2001, in International Patent OrganismDepositary National Institute of Advanced Industrial Science andTechnology, Tsukuba Central 6, 1-1, Higashi 1-Chome Tsukuba-shi,Ibaraki-ken, 305-8566, Japan. The present inventors also depositedArthrobacter ramosus S1 (FERM BP-7592), described in the presentspecification, on May 16, 2001, in International Patent OrganismDepositary National Institute of Advanced Industrial Science andTechnology, Tsukuba Central 6, 1-1, Higashi 1-Chome Tsukuba-shi,Ibaraki-ken, 305-8566, Japan.

INDUSTRIAL APPLICABILITY

[0131] As described above, the derivatives of cyclotetrasaccharide ofthe present invention are quite novel compounds, having substituentswhich could not be obtained by the conventional enzymatic reactionsystems, and which are synthesized by reacting reactive reagents withcyclotetrasaccharide. Therefore, since the physical properties ofcyclotetrasaccharide can be freely changed by the modification, noveluses of cyclotetrasaccharide, which has been impossible, can beprovided. Therefor, the derivatives of cyclotetrasaccharide of thepresent invention can be used intact as detergents, moisture-retainingagents, and skin-care agents by incorporating into foods, cosmetics, andpharmaceuticals. Also, novel organic compounds can be obtained bybinding cyclotetrasaccharide with the same or other compounds. Physicalproperties of cyclotetrasaccharide could be imparted to other compoundsby binding cyclotetrasaccharide with them.

1. A derivative of cyclic tetrasaccharide, which has a structurerepresented by Formula 1: Formula 1

wherein R₁ to R₁₂ mean optional substituents, but one or more of themare those except hydroxyl group and O-glycosyl group.
 2. The derivativeof cyclic tetrasaccharide of claim 1, where one or more substituents inR₁ to R₁₂ in Formula 1 are one or more members selected from the groupconsisting of hydrocarbon group, and substutuents having oxygen excepthydroxyl group and O-glycosyl group, nitrogen, sulfur, and halogen. 3.The derivative of cyclic tetrasaccharide of claim 1 or 2, which has anaverage degree of substitution of one or more.
 4. A process forproducing a derivative of cyclic tetrasaccharide, which comprises thesteps of allowing a reactive reagent to act on a compound represented byChemical formula 1, and substituting one or more substituents in R₁ toR₁₂ in Formula 1 with substituents except hydroxyl group and O-glycosylgroup: Chemical formula 1


5. The process of claim 4, wherein said compound represented by Chemicalformula 1 is in an anhydrous form.
 6. The process of claim 4 or 5,wherein said reactive reagent is one or more reagents selected from thegroup consisting of acids, bases, alcohols, aldehydes, ketones,halogens, amines, cyanogens, nitriles, oxyranes, isocyanates,isothiocyanates, thiols, sulfones, and their reactive derivatives. 7.The process of any one of claims 4 to 6, wherein said compoundrepresented by Chemical formula 1 is produced by allowing enzymes to acton starch.
 8. A composition comprising the derivative of cyclictetrasaccharide of any one of claims 1 to 3.